The Experts below are selected from a list of 1884 Experts worldwide ranked by ideXlab platform
Piet Herdewijn - One of the best experts on this subject based on the ideXlab platform.
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Synthesis of a Nucleobase-Modified ProTide Library
Organic letters, 2016Co-Authors: Ling-jie Gao, Steven De Jonghe, Piet HerdewijnAbstract:A new method for the construction of (aryloxy)Phosphoramidate nucleoside prodrugs is presented. An (aryloxy)Phosphoramidate ribose derivative as key building block was used for coupling with a number of nucleobases under Vorbruggen reaction conditions yielding the protected ProTides in excellent yields. Selective hydrolysis of the acetoxy groups on the sugar moiety afforded a series of the desired ProTides. The advantage of this approach, when compared to classical procedures, is the greater flexibility for achieving structural variety of the nucleobase moiety.
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aspartic acid based nucleoside Phosphoramidate prodrugs as potent inhibitors of hepatitis c virus replication
Organic and Biomolecular Chemistry, 2015Co-Authors: Munmun Maiti, Steven De Jonghe, Piet Herdewijn, Mohitosh Maiti, Jef RozenskiAbstract:In view of a persistent threat to mankind, the development of nucleotide-based prodrugs against hepatitis C virus (HCV) is considered as a constant effort in many medicinal chemistry groups. In an attempt to identify novel nucleoside Phosphoramidate analogues for improving the anti-HCV activity, we have explored, for the first time, aspartic acid (Asp) and iminodiacetic acid (IDA) esters as amidate counterparts by considering three 2′-C-methyl containing nucleosides, 2′-C-Me-cytidine, 2′-C-Me-uridine and 2′-C-Me-2′-fluoro-uridine. Synthesis of these analogues required protection for the vicinal diol functionality of the sugar moiety and the amino group of the cytidine nucleoside to regioselectively perform phosphorylation reaction at the 5′-hydroxyl group. Anti-HCV data demonstrate that the Asp-based Phosphoramidates are ∼550 fold more potent than the parent nucleosides. The inhibitory activity of the Asp-ProTides was higher than the Ala-ProTides, suggesting that Asp would be a potential amino acid candidate to be considered for developing novel antiviral prodrugs.
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iminodiacetic Phosphoramidates as metabolic prototypes for diversifying nucleic acid polymerization in vivo
Nucleic Acids Research, 2010Co-Authors: Anne Giraut, Xiaoping Song, Matheus Froeyen, Philippe Marliere, Piet HerdewijnAbstract:Previous studies in our laboratory proved that certain functional groups are able to mimic the pyrophosphate moiety and act as leaving groups in the enzymatic polymerization of deoxyribonucleic acids by HIV-1 reverse transcriptase. When the potential leaving group possesses two carboxylic acid moieties linked to the nucleoside via a Phosphoramidate bond, it is efficiently recognized by this error-prone enzyme, resulting in nucleotide incorporation into DNA. Here, we present a new efficient alternative leaving group, iminodiacetic acid, which displays enhanced kinetics and an enhanced elongation capacity compared to previous results obtained with amino acid deoxyadenosine Phosphoramidates. Iminodiacetic acid Phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) is processed by HIV-1 RT as a substrate for single nucleotide incorporation and displays a typical Michaelis–Menten kinetic profile. This novel substrate also proved to be successful in primer strand elongation of a seven-base template overhang. Modelling of this new substrate in the active site of the enzyme revealed that the interactions formed between the triphosphate moiety, magnesium ions and enzyme's residues could be different from those of the natural triphosphate substrate and is likely to involve additional amino acid residues. Preliminary testing for a potential metabolic accessibility lets us to envision its possible use in an orthogonal system for nucleic acid synthesis that would not influence or be influenced by genetic information from the outside.
Sergei M Gryaznov - One of the best experts on this subject based on the ideXlab platform.
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synthesis and properties of rna analogs oligoribonucleotide n3 p5 Phosphoramidates
Nucleic Acids Research, 1999Co-Authors: Tracy Matray, Sergei M GryaznovAbstract:The synthesis and characterization of RNA mimetics, uniformly modified oligoribonucleotide N3'-->P5' Phosphoramidates containing all four natural bases (uracil, cytosine, adenine and guanine) as well as thymidine and 2,6-diaminopurine, are described. These RNA analogs contain N3'-->P5' Phosphoramidate internucleotide linkages which replaced natural RNA O3'-->P5' phosphodiester groups. These oligonucleo-tides were constructed from novel monomeric units (2'- t -butyldimethylsilyl)-3'-(monomethoxyltrityl)-amino-nucleoside-5'- phos phoramidites, the preparation of which is also presented. Several mixed base 9-13mer oligoribonucleotide Phosphoramidates were synthesized with step-wise coupling yields of 96-98%. Thermal denaturation experiments demonstrated that ribo-N3'-->P5' Phosphoramidates form stable duplexes with a complementary RNA strand. Thus, the melting temperature ( T (m)) of a duplex formed by a 13mer ribo-N3'-->P5' Phosphoramidate (84 degrees C) was higher than that observed for the isosequential natural RNA oligomer (64.0 degrees C), or for the 2'-deoxy-N3'-->P5' Phosphoramidate counterpart (71.7 degrees C). Moreover, substitution of adenine by 2, 6-diaminopurine in an oligoriboPhosphoramidate pentamer resulted in a very significant increase in the duplex melting temperature ( approximately 7 degrees C per base substitution). The RNA Phosphoramidates also showed similar rates of hydrolysis by both RNase A and RNase T(1)as compared to natural RNA oligomers. The data presented indicate that this class of RNA analogs may be used as structural and functional RNA mimetics.
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rna mimetics oligoribonucleotide n3 p5 Phosphoramidates
Nucleic Acids Research, 1998Co-Authors: Sergei M Gryaznov, Holger WinterAbstract:The synthesis and properties of novel RNA mimetics, oligoribonucleotide N3'-->P5' Phosphoramidates, are described. These oligonucleotides contain 3'-aminoribonucleosides connected via N3'-->P5' Phosphoramidate linkages, replacing the native RNA O3'-->P5' phosphodiester counterparts. The key monomers 2'-t-butyldimethylsilyl-3'-(monomethoxytrityl)-amino-5'-phospho ramidi tes were synthesized and used to prepare the oligonucleotide Phosphoramidates using a solid phase methodology based on the phosphoramidite transfer reaction. OligoriboPhosphoramidates are very resistant to enzymatic hydrolysis by snake venom phosphodiesterase. These compounds form stable duplexes with complementary natural phosphodiester DNA and RNA strands, as well as with 2'-deoxy N3'-->P5' Phosphoramidates. The increase in melting temperature, Delta T m, was 5-14 degrees C relative to the 2'-deoxy Phosphoramidates for decanucleotides. Also, the thermal stability of the riboPhosphoramidatehomoduplex was noticeably higher (Delta T m +9.5 degrees C) than that for the isosequential 2'-deoxy Phosphoramidate complex. Furthermore, the oligopyrimidine ribo N3'-->P5' Phosphoramidate formed an extremely stable triplex with an oligopurine/oligopyrimidine DNA duplex with Delta T m +14.3 degrees C relative to the 2'-deoxy N3'-->P5' Phosphoramidate counterpart. The properties of the oligoribonucleotide N3'-->P5' Phosphoramidates indicate that these compounds can be used as hydrolytically stable structural and functional RNA mimetics.
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an oligodeoxyribonucleotide n3 p5 Phosphoramidate duplex forms an a type helix in solution
Nucleic Acids Research, 1996Co-Authors: Daoyuan Ding, Sergei M Gryaznov, David H Lloyd, S Chandrasekaran, Shijie Yao, L Ratmeyer, Yinquan Pan, David W WilsonAbstract:The solution conformations of the dinucleotide d(TT) and the modified duplex d(CGCGAATTCGCG)2 with N3'--> P5' Phosphoramidate internucleoside linkages have been studied using circular dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex conformation is similar to that of isosequential phosphodiester RNA, a A-type helix, and is different from that of DNA, a B-type helix, NMR studies of model dimers d(TpT) and N3'--> P5' Phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2'-endo to C3'-endo when the 3'-phosphoester is replaced by a Phosphoramidate group. Two-dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide Phosphoramidate and confirm that all furanose rings of 3'-aminonucleotides adopt predominantly N-type sugar puckering.
Christopher Mcguigan - One of the best experts on this subject based on the ideXlab platform.
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Synthesis and biological evaluation of 6-substituted-5-fluorouridine ProTides.
Bioorganic & medicinal chemistry, 2017Co-Authors: Magdalena Slusarczyk, Salvatore Ferla, Andrea Brancale, Christopher McguiganAbstract:A new family of thirteen Phosphoramidate prodrugs (ProTides) of different 6-substituted-5-fluorouridine nucleoside analogues were synthesized and evaluated as potential anticancer agents. In addition, antiviral activity against Chikungunya (CHIKV) virus was evaluated using a cytopathic effect inhibition assay. Although a carboxypeptidase Y assay supported a putative mechanism of activation of ProTides built on 5-fluorouridine with such C6-modifications, the Hint docking studies revealed a compromised substrate-activity for the Hint phosphoramidase-type enzyme that is likely responsible for Phosphoramidate bioactivation through P-N bond cleavage and free nucleoside 5'-monophosphate delivery. Our observations may support and explain to some extent the poor in vitro biological activity generally demonstrated by the series of 6-substituted-5-fluorouridine Phosphoramidates (ProTides) and will be of guidance for the design of novel Phosphoramidate prodrugs.
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application of Phosphoramidate pronucleotide technology to abacavir leads to a significant enhancement of antiviral potency
Journal of Medicinal Chemistry, 2005Co-Authors: Christopher Mcguigan, Susan Mary Daluge, Ed W Mclean, Harry Marr, Richard J Hazen, Kristjan Gudmundsson, T C Burnette, Sarah Harris, Lynn D Condreay, Lance C JohnsonAbstract:We report the first application of pronucleotide (ProTide) technology to the antiviral agent abacavir (Ziagen), used for the treatment of HIV infection. The phenylmethoxyalaninyl Phosphoramidate of abacavir was prepared in good yield in one step. Also prepared was the corresponding Phosphoramidate of the guanine nucleoside analogue “carbovir”. The antiviral profile of each of the parent nucleosides was compared to that of the Phosphoramidate ProTides. A significant (28- to 60-fold) increase in anti-HIV potency was noted for the ProTide of abacavir but not for that of carbovir. These findings were in agreement with the markedly higher (ca. 37-fold) levels of carbovir triphosphate that are formed in CEM cells upon response to the abacavir ProTide compared with the parent abacavir compound. In contrast the anti-HBV potency of both abacavir and carbovir were improved (10- and 20-fold, respectively) by ProTide formation. As in CEM cells, the abacavir ProTide provided significantly enhanced carbovir triphosphat...
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Phosphoramidate protides of 2 3 dideoxy 3 fluoroadenosine and related nucleosides with potent activity against hiv and hbv
Nucleosides Nucleotides & Nucleic Acids, 2003Co-Authors: Kristjan S Gudmundsson, Susan Mary Daluge, Richard J Hazen, Lance C Johnson, Lynn D Condreay, R Jansen, Christopher McguiganAbstract:Abstract Syntheses of Phosphoramidate protides of several 2′,3′-dideoxy-3′-fluoroadenosine derivatives by treatment of the nucleoside with phosphorochloridates in the presence of pyridine and t-BuMgCl is described. Several of these protides showed significantly improved antiviral potency over the parent nucleoside against HIV and HBV. Especially marked was the improvement in potency of Phosphoramidate protides of 2′,3′-dideoxy-3′-fluoroadenosine against both HIV and HBV.
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characterization of the activation pathway of Phosphoramidate triester prodrugs of stavudine and zidovudine
Molecular Pharmacology, 1999Co-Authors: D Saboulard, Christopher Mcguigan, Antonio Salgado, E De Clercq, Lieve Naesens, Dominique Cahard, R Pathirana, Sonsoles Velazquez, Jan BalzariniAbstract:The Phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although Phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.
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novel nucleoside Phosphoramidates as inhibitors of hiv studies on the stereochemical requirements of the Phosphoramidate amino acid
Antiviral Chemistry & Chemotherapy, 1996Co-Authors: Christopher Mcguigan, Antonio Salgado, C Yarnold, T Y Harries, E De Clercq, Jan BalzariniAbstract:Novel Phosphoramidate derivatives of the anti-HIV nucleoside analogue d4T were designed to act as labile membrane-soluble prodrugs of the bio-active free nucleotide d4TMP. We herein reveal the very marked dependence of the antiviral activity of these Phosphoramidates upon the stereochemistry of the amino acid attached to the phosphate centre; with a strong preference for the L-stereochemistry. These phosphate triesters were shown to liberate amino acid derivatives of the nucleotide intracellularly. These novel analogues, typified by alaninyl d4T monophosphate, may act as intracellular sources of the free nucleotides. The alaninyl d4T adducts themselves exert an antiviral effect when administered extracellularly, but again with clear distinctions between the L- and D-series. This evidence indicates that extracellularly administered blocked triesters derived from L-amino acids can generate d4TMP intracellularly, by a new pathway which is highly dependent on the amino acid stereochemistry.
Michael J Sofia - One of the best experts on this subject based on the ideXlab platform.
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Phosphoramidate prodrugs of β d 2r 4r dioxolane thymine dot as potent anti hiv agents
Antiviral Chemistry & Chemotherapy, 2012Co-Authors: Peiyuan Wang, Bruce S Ross, Suguna Rachakonda, Veronique Zennou, Meg Keilman, Congrong Niu, Donghui Bao, Phillip A Furman, Michael J Otto, Michael J SofiaAbstract:Background Nucleoside reverse transcriptase inhibitors (NRTIs) are an effective class of agents that has played a vital role in the treatment of HIV infections. (-)-β-D-(2R,4R)-dioxolane-thymine (DOT) is a thymidine analogue that is active against wild-type and NRTI-resistant HIV-1 mutants. It has been shown that the anti-HIV activity of DOT is limited due to poor monophosphorylation. Methods To further enhance the anti-HIV activity of DOT, an extensive structure-activity relationship analysis of Phosphoramidate prodrugs of DOT monophosphate was undertaken. These prodrugs were evaluated for anti-HIV activity using Hela CD4 β-gal reporter cells (P4-CCR5 luc cells). Results Among the synthesized prodrugs, the 4-bromophenyl benzyloxy l-alanyl phosphate derivative of DOT was the most potent, with a 50% effective concentration of 0.089 μM corresponding to a 75-fold increase in activity relative to the parent nucleoside DOT with no increased cytotoxicity. The metabolic stability of a selected number of potent DOT Phosphoramidates was also evaluated in simulated gastric fluid, simulated intestinal fluid, human plasma and liver S9 fractions. Conclusions A series of new Phosphoramidate prodrugs of DOT were prepared and evaluated as inhibitors of HIV replication in vitro. Metabolic stability studies indicated that these DOT Phosphoramidate derivatives have the potential to show acceptable stability in the gastrointestinal tract, but they metabolize rapidly in the liver.
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synthesis of diastereomerically pure nucleotide Phosphoramidates
Journal of Organic Chemistry, 2011Co-Authors: Bruce S Ross, Ganapati P Reddy, Hairen Zhang, Suguna Rachakonda, Michael J SofiaAbstract:Prodrugs of therapeutic nucleoside monophosphates masked as Phosphoramidate derivatives have become an increasingly important class of antiviral drugs in pharmaceutical research for delivering nucleotides in vitro and in vivo. Conventionally, Phosphoramidate derivatives are prepared as a mixture of two diastereomers. We report a class of stable phosphoramidating reagents containing an amino acid ester and two phenolic groups, one unsubstituted and the other with electron-withdrawing substituents. The reagents can be isolated as single diastereomers and reacted with the 5′-hydroxyl group of nucleosides through selective nucleophilic displacement of the substituted phenol to prepare single diastereomer Phosphoramidate products. This method has been used to prepare the HCV clinical candidate PSI-7977 in high yield and high diastereomeric purity.
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discovery of a β d 2 deoxy 2 α fluoro 2 β c methyluridine nucleotide prodrug psi 7977 for the treatment of hepatitis c virus
Journal of Medicinal Chemistry, 2010Co-Authors: Michael J Sofia, Bruce S Ross, Ganapati P Reddy, Hairen Zhang, Suguna Rachakonda, Peiyuan Wang, Donghui Bao, Wonsuk Chang, Dhanapalan Nagarathnam, Shalini BansalAbstract:Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the development of HCV therapies. β-d-2′-Deoxy-2′-α-fluoro-2′-β-C-methyl nucleosides are selective inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic. Phosphoramidate prodrugs of the 5′-phosphate derivative of the β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine nucleoside were prepared and showed significant potency in the HCV subgenomic replicon assay (<1 μM) and produced high levels of triphosphate 6 in primary hepatocytes and in the livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric mixture 14 was crystallized, and an X-ray structure was determined establishing the Phosphoramidate stereochemistry as Sp, thus correlating for the first time the stereochemistry of a Phosphoramidate prodrug with biological activity. 51 (PSI-7977...
Carston R Wagner - One of the best experts on this subject based on the ideXlab platform.
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Tunable Supramolecular Assemblies from Amphiphilic Nucleoside Phosphoramidate Nanofibers by Enzyme Activation
2018Co-Authors: Harrison T. West, Clifford M. Csizmar, Carston R WagnerAbstract:Enzymes possess unique qualities that make them ideal regulators of supramolecular assembly. They are uniquely sensitive to biomolecules and biological compartments, catalytic in effecting chemical reactions, and present a biocompatible and degradable platform for assembly regulation. We demonstrate the novel utility of Histidine Triad Nucleotide Binding Protein 1 (HINT1) in regulating supramolecular hydrogel formation. We synthesized nucleoside-Phosphoramidate-functionalized self-assembling peptides that we observed to form nanofibers. We found HINT1’s catalytic hydrolysis of the nucleoside Phosphoramidate moieties within the nanofiber structures to induce nanofiber organization and higher ordered assembly. The role of HINT1 in effecting this structural change was confirmed with experiments utilizing a high-affinity HINT1 inhibitor and catalytically dead HINT1 mutant. In addition, the kinetics and morphology of hydrogel formation were found to be dependent on the structure of the released nucleoside monophosphate. This work highlights the self-assembly of Phosphoramidate nanofibers and their higher organization triggered by HINT1 enzymatic activity
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31p nmr and genetic analysis establish hint as the only escherchia coli purine nucleoside phosphoramidase and as essential for growth under high salt conditions
Journal of Biological Chemistry, 2005Co-Authors: Tsuifen Chou, Pawel Bieganowski, Kara Shilinski, Jilin Cheng, Charles Brenner, Carston R WagnerAbstract:Eukaryotic cells encode AMP-lysine (AMP-N-epsilon-(N-alpha-acetyl lysine methyl ester) 5'-Phosphoramidate) hydrolases related to the rabbit histidine triad nucleotide-binding protein 1 (Hint1) sequence. Bacterial and archaeal cells have Hint homologs annotated in a variety of ways, but the enzymes have not been characterized, nor have phenotypes been described due to loss of enzymatic activity. We developed a quantitative (31)P NMR assay to determine whether Escherichia coli possesses an adenosine phosphoramidase activity. Indeed, soluble lysates prepared from wild-type laboratory E. coli exhibited activity on the model substrate adenosine 5'-monoPhosphoramidate (AMP-NH(2)). The E. coli Hint homolog, which had been comprehensively designated ycfF and is here named hinT, was cloned, overexpressed, purified, and characterized with respect to purine nucleoside Phosphoramidate substrates. Bacterial hinT was several times more active than human or rabbit Hint1 on five model substrates. In addition, bacterial and mammalian enzymes preferred guanosine versus adenosine Phosphoramidates as substrates. Analysis of the lysates from a constructed hinT knock-out strain of E. coli demonstrated that all of the cellular purine nucleoside phosphoramidase activity is due to hinT. Physiological analysis of this mutant revealed that the loss of hinT results in failure to grow in media containing 0.75 m KCl, 0.9 m NaCl, 0.5 m NaOAc, or 10 mm MnCl(2). Thus, cation-resistant bacterial cell growth may be dependent on the hydrolysis of adenylylated and/or guanylylated Phosphoramidate substrates by hinT.
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synthesis in vitro anti breast cancer activity and intracellular decomposition of amino acid methyl ester and alkyl amide Phosphoramidate monoesters of 3 azido 3 deoxythymidine azt
Journal of Medicinal Chemistry, 2000Co-Authors: Vidhya V Iyer, Edward J Mcintee, George W Griesgraber, Matthew R Radmer, Carston R WagnerAbstract:We report the synthesis and anticancer activity of a series of AZT Phosphoramidate monoesters containing amino acid methyl ester (3a−11a) and N-alkyl amide (3b−11b, 9c−9f) moieties. The aromatic amino acid methyl esters were found to be more cytotoxic than the aliphatic analogues toward MCF-7 cells (human pleural effusion breast adenocarcinoma cell line). A marked stereochemical preference for the l-amino acid stereochemistry was also observed in MCF-7 cells. There was no consistent enhancement of cytotoxicity of the methyl amides over the corresponding methyl esters. AZT and the two AZT aromatic amino acid methyl ester Phosphoramidates 8a and 9a were found to be more cytotoxic toward MCF-7 cells than to CEM cells (human T-cell lymphoblastic leukemia). The selective cytotoxicity toward MCF-7 cells may be associated with greater intracellular levels of Phosphoramidate monoester and/or phosphorylated AZT.
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synthesis and biological activity of aromatic amino acid Phosphoramidates of 5 fluoro 2 deoxyuridine and 1 β arabinofuranosylcytosine evidence of phosphoramidase activity
Journal of Medicinal Chemistry, 1996Co-Authors: Timothy W Abraham, Thomas I Kalman, Edward J Mcintee, Carston R WagnerAbstract:The amino acid Phosphoramidate diesters of FUdR (2) and Ara-C (6), 5-fluoro-2'-deoxy-5'-uridyl N-(1-carbomethoxy-2-phenylethyl)Phosphoramidate (5a), 5-fluoro-2'-deoxy-5'- uridyl N-(1-carbomethoxy-2-indolylethyl)Phosphoramidate (5b), 1-beta-arabinofuranosylcytosine 5'-N-(1-carbomethoxy-2-phenylethyl) Phosphoramidate (8a), and 1-beta-arabinofuranosylcytosine 5'-N-(1-carbomethoxy-2-indolylethyl)Phosphoramidate (8b), were synthesized and tested for their antitumor activity against L1210 mouse lymphocytic leukemia cells and CCRF-CEM human T-cell lymphoblastic leukemia cells. Ara-C Phosphoramidates 8a,b were found to be inactive at a concentration of 100 microM, while the FUdR conjugates 5a,b exhibited IC50 values within a range of 0.30-0.40 microM. Stability studies revealed that > 99% of the Phosphoramidates remained intact after incubation for > 2 days in 20% calf or 20% human serum. Intracellular thymidylate synthase (TS) inhibition studies revealed that treatment of L1210 and CCRF-CEM cells with 5a or 5b resulted in significant inhibition of TS in intact and permeabilized cells, while treatment of L929 TK- cells with these compounds did not result in inhibition of TS activity in intact cells. However, permeabilization of L929 TK- cells enhanced the activity of 5a,b toward intracellular TS by 900- and 1500-fold, respectively. In addition, incubation of cell-free extracts of CEM cells with radiolabeled 5b resulted in the rapid production of FUdR 5'-monophosphate and a lag in the generation of FUdR. Consequently, it is proposed that the metabolism of the Phosphoramidate diesters of FUdR in proliferating tissue proceeds through two separate enzymatic steps involving P-N bond cleavage by an unknown phosphoramidase followed by P-O bond cleavage by phosphatases such as 5'-nucleotidase.
